DNA mismatch repair is a major contributor to genetic stability. Mismatch repair defects confer strong cancer predisposition and have implications for cancer therapy because inactivation of the pathway renders cells resistant to the cytotoxic effects of certain anti-tumor drugs, a consequence of participation of the system in the DNA damage response. Perhaps surprisingly, mismatch repair function is also required for production of certain mutations, such as the expansion of (CAG){n} repeat sequences, the primary cause of a number of neurodegenerative diseases. The goals of this project to clarify conformations, conformational variation, and structures of multi-protein, protein-DNA, and multi-protein DNA assemblies that are key intermediates in DNA lesion processing and damage signaling by the mismatch repair system. Our aims are 3-fold: (1) Using deuterium exchange mass spectrometry (DXMS), we have identified regions of bacterial MutS and eukaryotic MutS-alpha (MSH2-MSH6) that undergo substrate-dependent conformational transitions. These regions will be subjected to site-directed mutagenesis and the resulting mutants characterized for their impact on mismatch repair both in vivo and in vitro. (2) Small angle X-ray scattering, equilibrium methods, DXMS, x-ray crystallographic, biochemical and genetic approaches as appropriate will be utilized to extend our understanding of multi-protein assemblies involved in mismatch repair. These assemblies will include PCNA complexes with MutS-alpha (MSH2-MSH6) and MutL-alpha (MLH1-PMS1/PMS2), MutSa complexes with exonuclease 1 and with Chk1, and the complex between exonuclease 1 and the BLM helicase. The latter study will be pursued in collaboration with Project 4. (3) Human MutS-alpha and MutS-Beta differ in the manner in which they interact with PCNA and MutL-alpha, indicating that MutS-alpha- and MutS-Beta-triggered repair events proceed by significantly different mechanisms. In view of the known involvement of MutS-Beta and MutL-alpha in the somatic phase (CAG){n}:(CTG){n}, triplet repeat expansion, we will seek small molecule inhibitors that specifically block MutS Beta-triggered repair events by screening for compounds that selectively block assembly the MutL-alpha-MutS-Beta-DNA ternary complex.